Light‐Controllable Binary Switch Activation of CAR T Cells

Abstract Major challenges to chimeric antigen receptor (CAR) T cell therapies include uncontrolled immune activity, off‐tumor toxicities and tumor heterogeneity. To overcome these challenges, we engineered CARs directed against small molecules. By conjugating the same small molecule to distinct tumor‐targeting antibodies, we show that small molecule specific‐CAR T cells can be redirected to different tumor antigens. Such binary switches allow control over the degree of CAR T cell activity and enables simultaneous targeting of multiple tumor‐associated antigens. We also demonstrate that ultraviolet light‐sensitive caging of small molecules blocks CAR T cell activation. Exposure to ultraviolet light, uncaged small molecules and restored CAR T cell‐mediated killing. Together, our data demonstrate that a light‐sensitive caging system enables an additional level of control over tumor cell killing, which could improve the therapeutic index of CAR T cell therapies.

Human primary T cells were activated with anti-CD3/CD28 beads (Life Technologies) with recombinant human IL-2 (Peprotech). Activated T cells were retrovirally transduced with CAR retrovirus supernatant in Retronectin (Takara)-coated plates. CAR expression was confirmed by flow cytometry using truncated CD19 marker.

Conjugation of small molecules to antibodies
Detailed method is described in supplemental information. Briefly, fluorescein was conjugated to anti-EGFR antibody (Cetuximab), anti-HER2 antibody (Pertuzumab), and anti-CD38 antibody (Daratumumab) by using NHS-

Cytotoxicity, activation and cytokine assays.
Target tumor cells were labeled with CellTrace dye violet or yellow (ThermoFisher Scientific) and TAA-specific antibodies conjugated with small molecules, and then 100,000 tumor cells were co-incubated with CAR T cells for 12-19 h at effector-to-target (E:T) ratio = 1:1. For unmasking of CMNB-caging group, cells were irradiated with 365nm UV light at 300 µW/m 2 for indicated time periods. Cells were collected and stained with Zombie NIR Fixable Viability dye and evaluated by flow cytometry analyses. Specific killing was calculated as (experimental death -spontaneous death)/(100 -spontaneous death) X 100%. Activation of CAR T cells was evaluated by coincubation with target cells at 1:1 ratio for 5 h. Cytokine production by CAR T cells was evaluated by coincubation with target tumor cells at 1:1 ratio for 12 h.
Statistics. Data presented as means ± SD. Results were analyzed by Student's t test, and statistical significance was calculated using a Student's t-test. P < 0.05 was considered significant and is designated with an asterisk in figures. Statistical analyses were done with Prism software (GraphPad Prism, RRID:SCR_002798).

Site-specific Conjugation via Glycan Remodeling
The concentrations of the peptides and proteins were determined using UV absorption at 280 nm and extinction coefficients based on the antibody's amino acid sequence. All aqueous solutions were prepared using Milli-Q water.
The reactions were performed as previously described [4] based on manufacturer's recommendation (Genovis, L1-

Determination of fluorescein-antibody ratio
The loading ratio was calculated based on the ratio of the absorption of fluorescein and antibody, as previously described in the literature. Briefly, the absorbance spectra of antibody-fluorescein conjugates were collected from 220 to 750 nm (NanoDrop spectrophotometer, ND-1000). The concentration of the chromophore fluorescein was calculated using the absorbance of the conjugates at 494 nm and extinction coefficient of 75,000 M -1 cm -1 , provided by Lumiprobe. The antibody concentration was estimated by subtracting fluorescein's contribution at 280 nm and the calculated extinction coefficient based on its amino acid sequence (217,440 M -for 10 min. For reducing and nonreducing gels, 4X reducing SDS sample buffer (Boston Bio Products, BP-110R) and 2X nonreducing SDS sample buffer (Bio-Rad, 1610737) were used, respectively. Second, the samples were loaded into 12% tris-tricine precast protein gels (Bio-Rad, 4561044) or 4-15% tris-glycine precast protein gels (Biorad, 5671083). Precision Plus Protein TM Dual Xtra Prestained Protein Standards (Bio-Rad, 1610377) were used for mass calibration. Electrophoresis was then performed at 200 V for 20 min. The gel was stained by Coomassie R250 and then destained using 10% acetic acid and 40% methanol. The gels were imaged using an iBright FL1000 Imaging system (Thermo Fisher Scientific). Figure S1.

Expression of small molecule-specific-CARs in T cell
Expression of FL-CAR and MPOB-CAR in primary human T cells.

Figure S2. Small molecule-specific CAR is as efficient as conventional CAR in tumor cell killing
Cytotoxicity of FL-CAR and conventional EGFR-CAR T cells were compared. MDA-BM-468 cells were labeled with a-EGFR or a-EGFR-FL and were co-cultured with FL-CAR or EGFR-CAR T cells (n=3). P values were determined by unpaired Student's t test. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure S10. Redirectable small molecule specific-CAR system against various types of targets and tumors
Cancer cell lines were labeled with naked or fluorescein-conjugated antibodies specific to each target, and cytotoxicity of FL-CAR T cells were tested after coculture with these labeled cancer cell lines (n=3). P values were determined by unpaired Student's t test. ****P < 0.0001.